Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8

Abstract

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial dihaem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-tobiomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a s 54 -dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria. INTRODUCTIONA surface-associated cytochrome c peroxidase (SACCP) isolated from the methanotrophic bacterium Methylococcus capsulatus (Bath) has recently been described (Karlsen et al., 2005). This protein shares significant sequence similarity with the members of the bacterial di-haem cytochrome c peroxidase family of proteins (BCCP, pfam03150) and appears to have a core structure resembling the resolved structures available from this family (Karlsen et al., 2005). However, the M. capsulatus SACCP can be distinguished from the other members of the BCCP family by being a much larger protein when compared to both the di-haem cytochrome c peroxidases and the MauG proteins, which constitute the two subfamilies (Chistoserdov et al., 1994;Ronnberg & Ellfolk, 1979). Furthermore, the two c-type haem-binding motifs of the M. capsulatus SACCP have, in their primary sequence, an increased inter-haem distance.However, structural analyses (Karlsen et al., 2005) strongly suggests that these haem groups in the native protein are structurally located in close proximity to each other, as previously demonstrated for other di-haem cytochrome c peroxidases (Fulop et al., 1995;Shimizu et al., 2001). We have suggested that the M. capsulatus SACCP protein is included in a third, novel subfamily of the BCCP family of proteins (Karlsen et al., 2005). In contrast to the di-haem cytochrome c peroxidases and the MauGs, which are localized in the periplasm (Goodhew et al., 1990; Wang et al., 2003), the M. capsulatus SACCP was isolated from the bacterial cell surface (Karlsen et al., 2005). Copperregulated multiple c-type cytochrom...

CorA Is a Copper Repressible Surface-Associated Copper(I)-Binding Protein Produced in Methylomicrobium album BG8

“…A corresponding protein immunoblot using antibodies recognizing the CorA co-transcribed c-type heme protein, CorB, showed, in line with previous data, the copper-regulated synthesis of CorB and confirmed that the corA-corB operon was transcribed under the low copper-to-biomass growth condition (Fig. 1b) [28]. Inspection of the Triton X-100 insoluble fractions from the low-copper grown cells revealed an abundant copper-repressible polypeptide of approximately 26 kDa (Fig.…”

“…To explore the cellular localization of CorA, low- and high-copper grown M. album BG8 cells were fractionated using the protocol previously applied to both M. capsulatus Bath and M. album BG8 (M&M and [28], [29]). The protein composition in the resulting fractions, including the soluble fraction (cytoplasm and periplasm), the Triton X-100 soluble fraction (inner membranes), and the Triton X-100 insoluble fraction (outer membranes) were assessed by SDS-PAGE (Fig.…”

“…Fractions enriched in soluble proteins, inner-membrane proteins, and outer-membrane proteins were obtained as described previously by Karlsen et al [28], [29].…”

“…Cell-surface-associated proteins and outer-membrane-associated proteins were extracted with NaCl as described previously [28], [29].…”

Chemistry and Biology of the Copper Chelator Methanobactin

“…47 Nearby genes encode a mutator type transposase (MettrDRAFT_3889) on the reverse strand and a neighboring hypothetical protein (MettrDRAFT_3890) on the forward strand, a sigma factor and FecR pair (MettrDRAFT_3891 and MettrDRAFT_3892), a TonB-dependent transporter (MettrDRAFT_3893), several unidentified and hypothetical proteins (MettrDRAFT_3894–3896), a member of the multidrug and toxic compound extrusion (MATE) family (MettrDRAFT_3897), an aminotransferase (MettrDRAFT_3898), a diheme enzyme (MettrDRAFT_3900) predicted to be a cytochrome c peroxidase and related to CorB from M. album BG8, 55 and another gene product probably associated with the diheme enzyme (MettrDRAFT_3899) (Table 1). A sigma factor (MettrDRAFT_3901) and the unknown protein that follows it (MettrDRAFT_3902) are the last consecutive coding regions found on the forward strand.…”